Applications , 7. IEX is a method for purification of proteins based on ionic interactions between proteins and surface charges opposite to those of the charged groups on an ionic resin see Figure 7. In cation-exchange chromatography, positively charged molecules are attracted to a negatively charged solid support.
Conversely, in anion-exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. The separation occurs due to competition between proteins with different charges on an ion-exchange resin. Proteins are complex ampholytes whose charges depend on the proportions of the amino acid residues in their structure as well as the acidity of the aqueous separation media.
The isoelectric point pI of a protein depends on the structural proportion of ionizable amino acids. Positive charges are typically found when the pH of the protein solution is below 8, due to the N-terminal amine and basic residues such as arginines, lysines, and histidines.
Similarly, negative protein charges typically exist above pH 6 and are due to the C-terminal carboxyl group and acidic e. The charged groups are almost always on the surface of proteins, except in case of metalloproteins, where the metal ion usually inside the molecule is usually coordinated by ligands amino acid residues of the protein . A stoichiometric model describes the relationship between the charged groups in a protein and the stationary phase.
The number of charged groups of the protein binds to the same number of oppositely charged groups of an ionic exchanger, and counterions are released both from the protein and the ion exchanger see Figure 7.
Protein retention on an ionic surface depends on the protein charge, surface charge, and the charge characteristics of the surrounding medium.
To describe this phenomenon, Kopaciewicz et al. Generally, anion-exchange chromatography AEX is used at pH values above the isoelectric point of the protein of interest, while cation-exchange chromatography CEX is performed below the isoelectric point.
At low pH or even at very high ionic strength, proteins may adsorb very strongly to an ion exchanger. This probably occurs due to an increase in the number of hydrogen bonds . The interaction between a protein and an ion exchanger depends not only on the net charge and the ionic strength but also on the surface charge distribution and conformation of the protein. Some structural changes can affect the separation by IEX. Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales.
The properties of the ion exchanger also influence the protein separation. Depending on the functional group, ion exchangers are classified as weak or strong. These ion exchangers are applicable in case of weakly ionizable proteins. The benefits of the use of weak ion exchangers are related to a reduced tendency for sample denaturation, less ability to bind impurities, as well as enhanced resolution.
In general, two methods are applicable in the elution strategy in IEX: The ion exchangers are classified as weak-cation, strong-cation, weak-anion, and strong-anion exchangers, respectively.
This method is based on making a step change in ionic strength of buffer solutions with the same pH in the ion exchange columns and can be used for identification and determination of the type of ion exchange groups on all sorts of ion exchangers see Figure 7. This investigation resulted in the observations that, after the step change from Tris-HCl buffer to Tris-HCl buffer with sodium chloride: In practice, the strategy based on the ionic strength by the addition of NaCl is the method of choice.
As a rule, more weakly charged proteins are eluted at lower salt concentrations, while the more strongly charged proteins are eluted at higher salt concentrations. Stepwise elution is often used for recovery of a concentrated protein especially in preparative chromatography. In this case, the optimization of gradient conditions is needed . Finally, IEX is one of the most used separation techniques in protein purification, for an advantage of this technique is that the elution normally takes place under mild conditions, and the protein can maintain its native conformation during the chromatographic process.
Limited selectivity is the major disadvantage of this method . To establish the structural and functional relationships in the characterization of all human proteins, of greatest importance are proteins of the blood. In proteomics, characterization of the blood proteome requires extensive fractionation prior to mass spectrometry analyses, and the removal of high-abundance proteins and subsequent enrichment of low-abundance proteins is a crucial step.
For this purpose, IEX can be combined with other chromatographic methods, mostly with SEC and chromatography on hydrophobic resins [21,22]. As shown in Figure 7. After the initial fractionation steps using ammonium sulfate precipitation and SEC, in the next steps, cation- and anion-exchange chromatography resulted in the highest number of identified proteins in the human plasma proteome .
In proteomics, multidimensional chromatographic separation and analysis of the proteins are performed at the peptide level, after proteolytic digestion of the entire proteins extracted from tissue samples the bottom-up approach . Strong cation-exchange chromatography SCEX is one of the frequently used liquid-chromatography strategies, where it has been shown that peptides are eluted according to their charge in a defined process.